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1 H Fast Field Cycling NMR (FFC-NMR) relaxometry is proposed as a powerful method to investigate tumour stroma in vivo upon the administration of a Gd-based contrast agent. To perform this study, an FFC-NMR equipment endowed with a wide bore magnet was used for the acquisition of Nuclear Magnetic Resonance Dispersion profiles on healthy muscle and tumour tissue in living mice. At magnetic field strengths < of ca. 1 MHz, the differences in the relaxation rates of the intra and extracellular compartment become of the same order of magnitude of the exchange rate across the cellular membranes. Under this condition, the water exchange rate between the two compartments yields to a biexponential magnetization recovery that can be analysed by fitting the experimental data with the two-Site eXchange (2SX) model. Using this model, it was possible to obtain, for the two compartments, both relaxation properties and water kinetic constants for water exchange across cell membranes. The method allowed us to determine the effect of the "matrix" on the water proton relaxation times and, in turn, to get some insights of the composition of this compartment, till now, largely unknown., (© 2019 John Wiley & Sons, Ltd.)

An innovative molecule, GdBLDL, for boron neutron capture therapy (BNCT) has been developed and its effectiveness as a BNCT carrier is currently under evaluation using in vivo experiments on small animal tumour models. The molecule contains both (10)B (the most commonly used NCT agent) and (157)Gd nuclei. (157)Gd is the second most studied element to perform NCT, mainly thanks to its high cross section for the capture of low-energy neutrons. The main drawback of (157)Gd neutron capture reaction is the very short range and low-energy secondary charged particles (Auger electrons), which requires (157)Gd to be very close to the cellular DNA to have an appreciable biological effect. Treatment doses were calculated by Monte Carlo simulations to ensure the optimised tumour irradiation and the sparing of the healthy organs of the irradiated animals. The enhancement of the absorbed dose due to the simultaneous presence of (10)B and (157)Gd in the experimental set-up was calculated and the advantage introduced by the presence of (157)Gd was discussed., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This study aims at developing an innovative theranostic approach for lung tumor and metastases treatment, based on Boron Neutron Capture Therapy (BNCT). It relies on to the use of low density lipoproteins (LDL) as carriers able to maximize the selective uptake of boron atoms in tumor cells and, at the same time, to quantify the in vivo boron distribution by magnetic resonance imaging (MRI). Tumor cells uptake was initially assessed by ICP-MS and MRI on four types of tumor (TUBO, B16-F10, MCF-7, A549) and one healthy (N-MUG) cell lines. Lung metastases were generated by intravenous injection of a Her2+ breast cancer cell line (i.e. TUBO) in BALB/c mice and transgenic EML4-ALK mice were used as primary tumor model. After neutron irradiation, tumor growth was followed for 30-40 days by MRI. Tumor masses of boron treated mice increased markedly slowly than the control group. From the clinical editor: In this article, the authors described an improvement to existing boron neutron capture therapy. The dual MRI/BNCT agent, carried by LDLs, was able to maximize the selective uptake of boron in tumor cells, and, at the same time, quantify boron distribution in tumor and in other tissues using MRI. Subsequent in vitro and in vivo experiments showed tumor cell killing after neutron irradiation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this study, the Huisgen reaction has been used to functionalise a carborane cage with a lipophilic moiety and a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand to obtain a new Gd boron neutron-capture therapy (BNCT)/magnetic resonance imaging (MRI) agent. The introduction of the triazole units has been accomplished under both heterogeneous conditions, by the use of a Cu-supported ionic-liquid catalyst, and homogeneous conditions. The ability of the Gd complex of the synthesised ligand to form stable adducts with low-density lipoproteins (LDLs) has been evaluated and then MRI has been performed on tumour melanoma cells incubated in the presence of a Gd-complex/LDL imaging probe. It has been concluded that the high amount of intracellular boron necessary to perform BNCT can be reached even in the presence of a relatively low-boron-containing LDL concentration., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tumor cells were targeted with Gd-loaded/LDL (low density lipoproteins) adducts consisting of ca 300 Gd(III) amphiphilic complexes incorporated in the lipophilic LDL particles. The long reorientational time of the Gd(III) complex in the supramolecular adduct yielded a relaxivity peak at ca 1 T, whereas its relaxivity at 7 T was 5 times less. The field-dependent relaxivity markedly affected the signal enhancement attainable at the two magnetic fields. As tumor cells showed up-regulation of LDL transporters, B16 melanoma cells were labeled with the Gd-loaded/LDL adduct. Each cell contained ca 2 × 10(9) Gd atoms. Upon dispersion of 5000 labeled cells in 1 μl of agar, signal intensity (SI) enhancements of about 30 and 7% were observed at 1 and 7 T, respectively. The results obtained on cellular systems were confirmed in vivo upon the administration of Gd-loaded/LDL particles to C57 mice bearing a transplanted melanoma (B16) tumor. From the herein reported results, one may conclude that, for slowly moving Gd complexes, it is possible to obtain in vivo sensitivity enhancements at 1 T several times higher than that attained at high fields., (Copyright © 2011 John Wiley & Sons, Ltd.)

The upregulation of low-density lipoprotein (LDL) transporters in tumour cells has been exploited to deliver a sufficient amount of gadolinium/boron/ligand (Gd/B/L) probes for neutron capture therapy, a binary chemio-radiotherapy for cancer treatment. The Gd/B/L probe consists of a carborane unit (ten B atoms) bearing an aliphatic chain on one side (to bind LDL particles), and a Gd(III)/1,4,7,10-tetraazacyclododecane monoamide complex on the other (for detection by magnetic resonance imaging (MRI)). Up to 190 Gd/B/L probes were loaded per LDL particle. The uptake from tumour cells was initially assessed on cell cultures of human hepatoma (HepG2), murine melanoma (B16), and human glioblastoma (U87). The MRI assessment of the amount of Gd/B/L taken up by tumour cells was validated by inductively coupled plasma-mass-spectrometric measurements of the Gd and B content. Measurements were undertaken in vivo on mice bearing tumours in which B16 tumour cells were inoculated at the base of the neck. From the acquisition of magnetic resonance images, it was established that after 4-6 hours from the administration of the Gd/B/L-LDL particles (0.1 and 1 mmol kg(-1) of Gd and (10)B, respectively) the amount of boron taken up in the tumour region is above the threshold required for successful NCT treatment. After neutron irradiation, tumour growth was followed for 20 days by MRI. The group of treated mice showed markedly lower tumour growth with respect to the control group., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To design efficient targeting strategies in magnetic resonance (MR) molecular imaging applications, the formation of supramolecular adducts between (strept)avidin ((S)Av) and tribiotinylated Gd-DOTA-monoamide complexes (DOTA=1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was explored. Two compounds based on the trivalent core of tris(2-aminoethyl)amine each containing three biotin molecules and one (L1) or three (L2) DOTA-monoamide (DOTAMA) ligands were synthesized. In these tribiotinylated derivatives the biotins are spaced far enough apart to allow the formation of the supramolecular adduct with the protein and to host the chelating units in between the (S)Av layers. Size exclusion HPLC analyses indicated complete formation of very high molecular weight polymers (>2 MDa) with (S)Av in solution. A (1)H NMR spectroscopy relaxometric study on the obtained polymeric adducts showed a marked increase of the relaxivity at 35-40 MHz as a consequence of the lengthening of the tumbling time due to the formation of Gd-chelates/(S)Av polymers. The most efficient Gd(3)L2/(S)Av polymeric system was used for a test in cell cultures. The target is represented by a neural cell adhesion molecule (NCAM), which is overexpressed in Kaposi's sarcoma cells and tumor endothelial cells (TEC) and that is efficiently recognized by a biotinylated tetrameric peptide (C3d-Bio). In vitro experiments showed that only cells incubated with both C3d-Bio and Gd(3)L2/SAv polymer were hyperintense with respect to the control. Relaxation rates of cell pellets incubated with Gd(3)L2/SAv alone were not significantly different from the untreated cells demonstrating the absence of a specific binding.

There is an ongoing desire to produce high-relaxivity, Gd-based magnetic resonance imaging (MRI) contrast agents. These may allow for lower doses to be used, which is especially important in view of the current safety concerns surrounding Gd in patients. Here we report the synthesis of a high-relaxivity MRI contrast agent, by incorporating Gd-chelating lipids that coordinate two water molecules into high-density lipoprotein (q = 2 HDL). We compared the properties of q = 2 HDL with those of an analogous HDL particle labeled with Gd-chelating lipids that coordinate only one water molecule (q = 1 HDL). We found that the q = 2 HDL possessed an elevated r(1) of 41 mM(-1) s(-1) compared to 9 mM(-1) s(-1) for q = 1 HDL at 20 MHz, but the q = 2 HDL exhibited high R(2)* values at high fields, precluding imaging above 128 MHz. While carrying out this investigation we observed that enlarged, disrupted particles were formed when the synthesis was carried out above the lipid critical micelle concentration (cmc), indicating the importance of synthesis below the cmc when modifying lipoproteins in this manner. The high relaxivity of q = 2 HDL means it will be an efficacious contrast agent for future MR imaging studies.

Tumor vessel imaging could be useful in identifying angiogenic blood vessels as well as being a potential predictive marker of antiangiogenic treatment response. We recently reported the expression of the neural cell adhesion molecule (NCAM) in the immature and tumor endothelial cell (TEC) lining vessels of human carcinomas. Exploiting an in vivo model of human tumor angiogenesis obtained by implantation of TEC in Matrigel in severe combined immunodeficiency mice, we aimed to image angiogenesis by detecting the expression of NCAM with magnetic resonance imaging. The imaging procedure consisted of (a) targeting NCAMs with a biotinylated derivative of C3d peptide that is known to have high affinity for these epitopes and (b) delivery of a streptavidin/gadolinium (Gd)-loaded apoferritin 1:1 adduct at the biotinylated target sites. The remarkable relaxation enhancement ability of the Gd-loaded apoferritin system allowed the visualization of TEC both in vitro and in vivo when organized in microvessels connected to the mouse vasculature. Gd-loaded apoferritin displayed good in vivo stability and tolerability. The procedure reported herein may be easily extended to the magnetic resonance visualization of other epitopes suitably targeted by proper biotinylated vectors.

The glutamine transporting system is up-regulated in tumor cells because cell proliferation requires the uptake of large quantities of glutamine. It has been found that the paramagnetic magnetic resonance imaging (MRI) reporter Gd-DOTAMA-C6-Gln, where the glutamine residue is covalently bound to the Gd chelate through a C6 spacer, accumulates in tumor cells both "in vitro" and "in vivo" experiments. The observation that the relaxivity of cellular pellets does not increase with the increase in the amounts of entrapped Gd chelate is taken as an indication that the internalization has occurred through receptor mediated endocytosis. The iv administration of Gd-DOTAMA-C6-Gln allowed the MRI visualization of tumor masses in A/J mice grafted with the murine neuroblastoma cell line Neuro-2a and in Her-2/neu transgenic mice developing multiple mammary carcinoma, respectively.

The high binding affinity between avidin and biotin has been exploited to develop a procedure for magnetic resonance imaging (MRI) visualization of target cells. SHIN3 and PANC1 tumor cell lines have been used as target cells because they possess on their membranes galactosyl receptors able to bind avidin molecules. Avidin-Gd chelate adducts have been built by using two Gd complexes containing one (Gd-I) and two (Gd-II) biotin residues, respectively. The relaxivities of such supramolecular adducts are significantly higher than those shown by free Gd-I and Gd-II. There is evidence of the occurrence of multilayered adducts in which the bis-biotinylated Gd(3+) complex acts as a bridge between adjacent avidin molecules. MRI differentiation of labeled versus unlabeled cells has been attained when approximately 6 x 10(8) Gd units were internalized in each cell. Furthermore, there is a marked decrease in the measured intracellular T(1) relaxivity as the number of internalized Gd complexes increases, probably owing to too short relaxation times of endosomic water protons with respect to their diffusion lifetime.

Five novel Gd(iii) complexes based on the structure of the heptadentate macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) ligand have been synthesized and their (1)H and (17)O NMR relaxometric properties investigated in detail. The complexes have been functionalised on the secondary nitrogen atom of the macrocyclic ring with different pendant groups for promoting their ability to interact non-covalently with human serum albumin (HSA). The analysis of the proton relaxivity, measured as a function of pH and magnetic field strength, have revealed that the three complexes bearing a poly(ethylene glycol)(PEG) chain possess a single coordinated water molecule, whereas the complexes functionalised with 1-[3-(2-hydroxyphenyl)]-propyl and 1-[3-(2-carboxyphenyloxy)]-propyl pendant groups have two inner sphere water molecules. The water exchange rates, measured by variable temperature (17)O NMR, cover a broad range of values (from 18 to 770 ns) as a function of their charge, the chemical nature of the substituent and its ability to organize a second sphere of hydration near the water(s) binding site. All the complexes have shown some degree of interaction with HSA, with a stronger binding affinity measured for those bearing an aromatic moiety on the pendant group. However, upon binding the expected relaxation enhancement has not been observed and this has been explained with the displacement of the coordinated water molecules by the protein and formation of ternary adducts.

Can gadolinium III [Gd(III)] complexes be considered good candidates for magnetic resonance (MR)-molecular imaging studies? In this review article, we examine the principal issues that are the basis of successful use of Gd-based protocols in molecular imaging applications. High relaxivity is the primary requisite. Therefore, the design of such paramagnetic probes has to be pursued keeping in mind the relationships between structure, dynamics, and the relevant parameters involved in paramagnetic relaxation processes. Moreover, the limited number of target molecules on cellular membranes makes it necessary to define strategies aimed at delivering many Gd-containing moieties to the sites of interest. Examples are reported for the attainment of very high relaxivities for the design of new routes for pursuing the accumulation of small sized Gd(III) complexes at the targeting sites. An efficient cellular uptake of Gd-containing probes is the key step for attaining the visualization of targeted cells by MR imaging, and selected examples are reported. In this context, the problem of the assessment of the minimum amount of Gd(III) complexes necessary for the MR imaging-visualization of cells has been addressed by reporting the authors' observations on the cell-internalization of Gd(III) complexes. A particularly efficient delivery system is represented by Gd-loaded apoferritin, which allows the MR visualization of hepatocytes when the number of Gd-complexes per cell is 4 +/- 1 x 10(7). Finally, the potential of responsive systems is considered by outlining the exploitation of the amplification effect brought about by the action of a specific enzymatic activity on the relaxivity of a suitably functionalized Gd(III) complex., (Copyright 2002 Wiley-Liss, Inc.)

A novel ligand based on a pyridine-containing macrocycle bearing two acetic and one methylenephosphonic arms (PCP2A) has been synthesized. An efficient synthesis of PCP2A is based on the macrocyclization reaction between 2,6-bis(chloromethyl)pyridine and a 1,4, 7-triazaheptane derivative bearing a methylenephosphonate group on N-4. The Gd(III) complex of PCP2A displays characteristic properties which make it a very promising contrast agent for improved applications in magnetic resonance imaging. In fact it shows (i) a very high stability constant (log K(GdPCP2A) = 23.4) which should guarantee against the in vivo release of toxic free Gd(III) ions and free ligand molecules and (ii) a relaxivity that is about 2 times higher than the values reported for contrast agents currently used in the clinical practice. Its high relaxivity is the result of the presence of two water molecules in the inner coordination sphere and a significant contribution from water molecule(s) hydrogen bonded to the phosphonate group. Moreover, the inner sphere water molecules are involved in an exchange with the bulk water which is relatively fast. This property is important for the attainment of an even higher relaxivity once the molecular reorientation rate of the [GdPCP2A(H(2)O)(2)](-) moiety is lengthened by means of conjugation to a macromolecular substrate.

Macromolecular Gd(III) complexes may find useful application as contrast agents for magnetic resonance angiography (MRA). Herein two novel systems are reported, namely Gd(DO3ASQ)3-lys16 and Gd(DO3ASQ)30-orn114. Their syntheses are based on the ability of the squaric acid moiety to act as a linker between the DO3A (1,4,7, 10-tetraazacyclododecane-1,4,7-triacetic acid) chelate moiety and the polyamino acidic chain. Moreover, the squaric acid participates in the coordination cage of the Gd(III) ion. The investigation of 1H and 17O NMR relaxation processes of solvent water nuclei allowed a detailed characterization of the systems under study. Gd(DO3ASQ)30-orn114 displays a remarkable ability to enhance the water proton relaxation rate of its solutions, and it may be considered as potential contrast agent for MRA applications.